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EPIGENETIC BASIS OF STEM CELL IDENTITY IN NORMAL AND MALIGNANT HEMATOPOIETIC DEVELOPMENT by Namyoung Jung A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July, 2015 © 2015 Namyoung Jung All Rights Reserved Abstract Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by subpopulations of leukemia-initiating or leukemia stem cells (LSC) that give rise to clonally related non-stem leukemic blasts. The LSC model proposes that since LSC and their blast progeny are clonally related, their functional properties must be due to epigenetic differences. In addition, the cell of origin of LSC among normal hematopoietic stem and progenitor cells (HSPCs) has yet to be clearly demonstrated. In order to investigate the role of epigenetics in LSC function and hematopoietic development, we profiled DNA methylation and gene expression of CD34+CD38-, CD34+CD38+ and CD34- cells from 15 AML patients, along with 6 well-defined HSPC populations from 5 normal bone marrows using Illumina Infinium HumanMethylation450 BeadChip and Affymetrix Human Genome U133 Plus 2.0 Array. To define LSC and blast functionally, we performed engraftment assays on the three subpopulations from 15 AML patients and defined 20 LSCs and 24 blast samples. We identified the key functional LSC epigenetic signature able to distinguish LSC from blasts that consisted of 84 differential methylations regions (DMRs) in 70 genes that correlated with differential gene expression. HOXA cluster genes were enriched within the LSC epigenetic signature. We found that most of these DMRs involve epigenetic alteration independent of underlying mutations, although several are downstream targets of genetic mutation in epigenome modifying enzymes and upstream regulators. The LSC epigenetic signature could predict overall survival for AML patients independent of known risk factors such as age and cytogenetics. We characterized epigenetic changes during normal human hematopoietic development and identified key novel regulators for ii hematopoietic differentiation such as HMHB1 and MIR539. We found that global hypomethylation is a critical mechanism of lineage commitment in human hematopoiesis. Our DNA methylation analysis in human hematopoiesis revealed variable epigenetic regulation compared to murine hematopoiesis. Furthermore, we found that LSC populations formed two distinct clusters resembling either lymphoid-primed multipotent progenitors (L-MPPs) or granulocyte/macrophage progenitors (GMPs). These results provide the first evidence for epigenetic variation between LSC and their blast progeny in AML, and its prognostic power. We also provided comprehensive methylome map of human hematopoiesis and identified epigenetically distinct subgroups of AML LSCs that likely reflect the cell of origin. Readers: Ravindra Majeti, M.D., Ph.D. Andrew P. Feinberg, M.D., M.P.H. Thesis Advisor: Andrew P. Feinberg, M.D., M.P.H. iii Acknowledgments Over the past 6 years of my graduate study, many people have contributed to my personal and professional growth. It would be impossible to mention everyone who helped me to finish this long journey in graduate school, but I wish to acknowledge couple people here. Foremost, I would like to express my deepest gratitude to my thesis advisor, Dr. Andrew Feinberg for his relentless support, patience, and motivation. Andy has given insightful suggestions and comments on any subject that we had a conversion on. He also taught me how I should behave in academic setting as a professional scientist. Without Andy’s guidance and persistent help throughout my graduate study, this thesis would not have been possible. I would like to thank to Dr. Ravindra Majeti at Stanford who has been a wonderful thesis committee member, the second reader of my thesis, and collaborator. Ravi has provided his expert advices and valuable knowledge in leukemia and hematopoiesis throughout this thesis. His comments and suggestions were an enormous help for me to learn knowledge in a different field. I am deeply grateful to my thesis committee chair, Dr. Roger Reeves for his generous support and encouragement for this thesis and my career, and thesis committee member, Dr. Donald Small for his advices and support. I wish to acknowledge Dr. Yunje Cho, my undergraduate thesis advisor, for his encouragements and advices, which have been motivation for my PhD work. iv I am deeply grateful to my collaborators, Dr. Andrew Gentles and Dr. Rafael Irizarry for their statistical advices and comments, and Dr. Bo Dai for the essential part of functional experiments of this thesis. I would like to express my gratitude toward the AML patients and their family members for their decisions to provide valuable samples for this thesis. I could not have done my PhD work without the advices and supports provided by current and former Feinberg lab members who have had impact on every aspect of this thesis. Akiko Doi and Brian Herb have always offered great feedbacks for experiments and encouragements to move forward during my graduate course work. It has been very lucky to have Amy Vandiver as my bench mate for past 5 years, who has provided useful suggestions for science, and warm support for any decision related to graduate course work and even personal life. I’m grateful to Carolina Montano, Lindsay Rizzardi, Hwajin Lee, Xin Li, Yun Liu, Hong Ji, Peter Murakami, Michael Multhaup, Varenka Rodriguez, and Elisabet Pujadas for useful discussions, feedbacks, and their friendship. I thank to Rakel Tryggvadottir and Arni Runarsson for their experimental help. I am grateful to Samsung Scholarship that has offered financial support for first five years, and Mogam Science Scholarship Foundation for financial support for last year of my graduate study. Many friends have supported and helped me to stay centered throughout past 6 years. I have been fortunate to have warmhearted CMM classmates, who have helped me to adapt to a new culture. Friends of Korean community and in Korea have been always there to listen to my problems, and given me great emotional support. v Last but not least, this long journey in graduate school would have been impossible without the warm support of my family. My grandparents, Jongsung Jung and Oksoon Cho have offered priceless life lessons and taught me how to be a polite person. My parents, Insoo Cheong and Geumsook Choi, have provided unconditional love and support for any decision that I have made. My two younger sisters, Gayoung Jung and Chaeyoung Jung have been the best friends throughout my life giving me emotional support. I will always be grateful to all the individuals mentioned above who shaped me as a professional scientist. vi Table of Contents Title Page ............................................................................................................................. i Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents .............................................................................................................. vii List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... xi Chapter 1: Introduction ........................................................................................................1 Chapter 2: Epigenetic signature of leukemia stem cell ......................................................27 Chapter 3: Epigenetic basis of human normal hematopoietic development ....................135 Chapter 4: The cell of origin of leukemia stem cell .........................................................168 References ........................................................................................................................190 Curriculum Vitae .............................................................................................................207 Appendix .................................................................................................... See attached files vii List of Tables Table 1.1. French-American-British (FAB) classification ................................................25 Table 1.2. Cytogenetics and prognosis ..............................................................................26 Table 2.1. Clinical features of AML patients in this study ...............................................60 Table 2.2. Genetic mutations identified ............................................................................61 Table 2.3. Engraftment of AML Subpopulations ..............................................................62 Table 2.4. DMRs of LSC vs Blast ............................................................... See Appendix1 Table 2.5. Summary of LSC vs Blast DMRs ....................................................................64 Table 2.6. LSC epigenetic signature .................................................................................65 Table 2.7. Second DMR analysis to examine confounding effect of MLL cases .............70 Table 2.8. Ingenuity pathway analysis ..............................................................................71
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